Standard protocols exist in the literature for generating double-stranded cDNAs from cellular mRNA. See, e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
Generally, to prepare a double stranded cDNA from RNA, a first cDNA strand is synthesized using RNA as a template, resulting in an mRNA:DNA hybrid. The mRNA is then degraded by enzymes such as RNAse H. The second cDNA strand is then synthesized by using the first cDNA strand as a template.
Many methods exist for the synthesis of the second strand, but these methods have disadvantages. One method involves self-priming. The 3′ termini of single-stranded cDNAs form hairpin structures that can be used to prime synthesis. In this method, the RNA strand of the hybrid is degraded, and the resulting hairpin loop that forms is utilized as a primer by Klenow fragment or RT to initiate synthesis of the second strand. The loop is then digested with the single-stranded-specific nuclease S1 to yield a double-stranded cDNA. A self-primed synthesis of the second strand is a poorly controlled reaction, and the subsequent cleavage of the hairpin structure can lead to the loss or rearrangement of sequences corresponding to the 5′ terminus of the mRNA.
Another method involves nick translation. RNAase H produces nicks and gaps in the RNA strand of the hybrid, creating a series of RNA primers that can be extended by E.coli DNA polymerase I. With nick-translation, the amount of RNAase H activity must be closely monitored and controlled; there must be sufficient hydrolysis of the RNA to generate large gaps, but too much degradation will result in few or no primers for synthesis. The length of the RNA primers is also important in terms of the melting temperature (Tm), and forming a stable complex between the primer and the DNA template. Further, the existence of a series of DNA fragments representing the second strand must be ligated to generate a full length second strand. The ligation reaction is often inefficient, especially when a gap exists between the two DNA fragments.
Another method employs primed synthesis using random primers or homopolymeric tailing. In random priming, random hexamers serve as primers for DNA synthesis by T4 DNA polymerase. This method requires the addition of T4 ligase and T4 polynucleotide kinase for ligation of the fragments to generate a continuous second strand. A problem with random hexamer priming is that the reaction is performed at 37° C., which is above the Tm for annealing of the hexamers to the DNA template. In addition, a strong sequence influence exists; GC-rich regions are primed at a higher frequency than AT-rich regions. In areas of the template where there is secondary structure, the hexamers may not anneal. Hence, secondary structure can have a strong influence as to where synthesis is initiated and terminated, which can result in an incomplete synthesis of the second strand. Furthermore, random priming requires the synthesis of the random primers on a DNA synthesizer followed by exogenous annealing, which can be burdensome.
In another method terminal transferase is used to add homopolymeric tails of dC residues to free 3′-hydroxyl groups in the first DNA strand. This tail is then hybridized to oligo(dG), which serves as a primer for the synthesis of the second strand. A problem with homopolymeric tailing by terminal transferase is that the reaction can not be closely controlled. The length of tails added can vary significantly as well as the number of RNA strands that get tailed. In addition, oligo(dG) and oligo(dG):poly(dC) can form stable secondary structures that can inhibit synthesis by “tying up” the primer in secondary structure and thus inhibiting its ability to prime synthesis.
Thus, there remains a need for a reliable method for preparing the second strand of cDNA.